Heterologous expression of 2-deoxyribose-5-phosphate aldolase and application of 2-deoxyribose-5-phosphate aldolase in catalytic synthesis of statin drug intermediates

Abstract

The invention belongs to the crossing field of bioengineering and pharmaceutical technology, and particularly relates to a heterologous expression technology of 2-deoxyribose-5-phosphate aldolase derived from rhodococcus ruber SD3 and optimization of aldol condensation reaction conditions of substrates acetaldehyde and chloroacetaldehyde catalyzed by the aldolase. The enzyme can catalyze a substrate to specifically synthesize a statin drug key intermediate (3R, 5S)-6-chloro-2, 4, 6-trideoxy pyranoside, the conversion rate can reach 44.45%, and a new biological catalysis approach and technical support are provided for efficient and green production of statin drugs. The rhodococcus ruber SD3 is preserved in the China Center for Type Culture Collection, and the preservation number of the rhodococcus ruber SD3 is CCTCC NO: M 2012035.

Description

Technical Field

[0001] This invention relates to the application of biocatalysis in the synthesis of pharmaceutical intermediates, specifically to the gene cloning and heterologous expression of 2-deoxyribose-5-phosphate aldolase (DeoC) derived from Rhodococcus SD3, and the enzyme-catalyzed synthesis of key intermediates for statin drugs. 3R , 5S The process conditions for 6-chloro-2,4,6-trideoxypyranoside belong to the interdisciplinary field of bioengineering and pharmaceutical chemistry. Background Technology

[0002] Statins are currently the first-line drugs for the clinical treatment of hypercholesterolemia and cardiovascular disease. They significantly reduce the level of low-density lipoprotein cholesterol in the blood by inhibiting HMG-CoA reductase, a key enzyme in the cholesterol synthesis pathway. 3R , 5S 6-Chloro-2,4,6-Trideoxypyranoside is a core chiral intermediate for the synthesis of next-generation potent statin drugs (such as pitavastatin and atorvastatin), and the efficient synthesis of this intermediate is a key step in the production of statin drugs.

[0003] Compared with traditional chemical synthesis methods, biocatalytic synthesis has many significant advantages. The reaction conditions are mild, avoiding the harsh conditions of high temperature and high pressure that place high demands on equipment and consume large amounts of energy, thus reducing production costs and environmental pollution, aligning with the development concept of green chemistry. Biocatalytic reactions exhibit high selectivity, reducing side reactions, improving product purity and yield, and lowering the cost and difficulty of subsequent separation and purification steps. This invention uncovers a novel catalytic activity of 2-deoxyribonucleotide-5-phosphate aldolase in Rhodococcus SD3, and explores a new method for synthesizing statin drug intermediates using this enzyme, providing a green and efficient route for drug synthesis.

[0004] Rhodococcus is a group of Gram-positive bacteria widely found in extreme environments such as soil and industrial wastewater. The enzymes synthesized intracellularly often possess unique properties such as strong stress resistance, broad substrate range, and high catalytic efficiency, making them an important source of novel industrial enzymes. Our laboratory previously screened and obtained a Rhodococcus SD3 strain, and subsequently successfully obtained [the desired enzyme] using genome cloning and sequencing technology. deoC The gene was cloned, heterologously expressed, and purified for application in the catalytic synthesis of statin drug intermediates, aiming to solve the technical bottlenecks of existing DeoC enzymes and promote the greening and efficiency of statin drug production. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the issue of using wild-type enzymes to catalyze the preparation of (…) 2-deoxy-D-ribose-5-phosphate aldolase mutants and their applications, as described in the previously reported patent (application number: CN202410267867.7). 3R , 5S 6-Chloro-2,4,6-Trideoxypyranoside suffers from low enzyme activity and poor substrate conversion, requiring semi-rational design and iterative mutagenesis to improve substrate conversion. This application provides a wild-type aldolase with high catalytic activity and strong substrate tolerance. The gene of this aldolase is cloned, a recombinant expression vector is constructed, and methods for preparing the aldolase and its application in catalytic synthesis are described. 3R , 5S Applications of 6-chloro-2,4,6-trideoxypyranoside. The preparation method of aldolase was simplified.

[0006] In order to solve the existing technical problems, the present invention provides a novel 2-deoxyribose-5-phosphate aldolase derived from Rhodococcus SD3, which is a protein composed of the amino acid sequence shown in SEQ ID No. 1.

[0007] The present invention also provides a gene encoding the 2-deoxyribose-5-phosphoaldolase, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0008] This invention provides a recombinant cloning vector and expression vector containing the 2-deoxyribose-5-phosphate aldolase gene.

[0009] This invention provides a method for preparing 2-deoxyribose-5-phosphate aldolase, the method comprising: (a) The expression vector is cultured under suitable expression conditions to express the 2-deoxyribose-5-phosphate aldolase; (b) Purify the expression product to obtain the recombinant protein of 2-deoxyribose-5-phosphate aldolase.

[0010] This invention provides a method for preparing statin drug intermediates using inexpensive and readily available chloroacetaldehyde and acetaldehyde under the catalysis of 2-deoxyribose-5-phosphate aldolase. 3R , 5S )-6-chloro-2,4,6-trideoxypyranoside, the synthetic pathway is shown below:

[0011] The buffer solution used in the catalytic process of this invention is a conventional buffer solution in the art, with a suitable pH of 4-9; more suitable buffer solutions are sodium citrate buffer (pH 4-6), phosphate buffer (pH 6-8), and Tris-HCl buffer (pH 8-9); the buffer solution with the optimal catalytic activity is phosphate buffer at pH 7.0.

[0012] The temperature conditions used in the catalytic process of this invention are 20℃-50℃, and the optimal temperature for catalytic activity is 30℃.

[0013] The chloroacetaldehyde concentration used in the catalytic process of this invention is 5 mM-100 mM, the acetaldehyde concentration is twice that of chloroacetaldehyde, and the optimal substrate concentration for catalytic activity is 25 mM. Attached Figure Description

[0014] Figure 1 for deoC Detection of gene amplification products and gel recovery results. A. PCR amplification of the target gene. deoC B. Target gene deoC Agarose gel recovery. Note: Lane M: DL2000 marker.

[0015] Figure 2 To detect the linearized product and gel recovery results of the reverse amplification of vector pET-28a. A. Reverse PCR linearized vector pET-28a(+); B. Agarose gel recovery of linearized vector pET-28a(+). Note: Lane M: DL10000 marker.

[0016] Figure 3 To verify the recombinant plasmid pET-28a(+)- by PCR amplification deoC Positive clone result.

[0017] Figure 4 This is an SDS-PAGE electrophoresis image of DeoC protein expression.

[0018] Figure 5 This is a diagram showing the purification of the recombinant protein DeoC by nickel column affinity chromatography.

[0019] Figure 6 For products ( 3R , 5S High-resolution mass spectrum of 6-chloro-2,4,6-trideoxypyranoside.

[0020] Figure 7 The difference in DeoC catalytic activity under different pH conditions.

[0021] Figure 8 Differences in DeoC tolerance under different pH conditions.

[0022] Figure 9 Differences in the catalytic activity of DeoC at different reaction temperatures.

[0023] Figure 10 Differences in the tolerance of DeoC at different temperatures.

[0024] Figure 11 Effect of different substrate concentrations on the catalytic activity of DeoC.

[0025] Figure 12 Chloroacetaldehyde concentration and OD 528 The linear relationship of nm.

[0026] This invention provides a method for preparing and using 2-deoxyribose-5-phosphate aldolase. Specifically, the 2-deoxyribose-5-phosphate aldolase gene from Rhodococcus SD3 is amplified, heterologously expressed, and purified. The aldolase is then used in the in vitro synthesis of intermediates for statin drugs. 3R , 5S 6-chloro-2,4,6-trideoxypyranoside. Based on this, the present invention was completed. Detailed Implementation

[0027] Example 1: Recombinant vector pET-28a(+)- deoC The construction of the enzyme and the preparation of the recombinase.

[0028] Using Rhodococcus SD3 genomic DNA as a template for PCR, forward primers (5'-agcaaatgggtcgcggatccATGCCGGAGACCACCCTCA-3') and reverse primers (5'-tcgagtgcggccgcaagcttTCACTCGGGCAGGCCGTC-3') were designed for PCR amplification. The PCR reaction was performed in a 50 μL system under the following conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 1 min, 58℃ annealing for 30 s, and 72℃ extension for 38 s, for a total of 30 cycles; and a final incubation at 72℃ for 10 min. The amplified products and gel-recovered products were detected by 1% agarose gel electrophoresis. The results are shown in the figure below. Figure 1Using the pET-28a(+) plasmid as a template for reverse PCR amplification, DNA amplification was performed using specific PCR primers (pET-28a(+)-F: AAGCTTGCGGCCGCACTC pET-28a(+)-R: GGATCCGCGACCCATTTG) to obtain the linearized cloning vector pET-28a(+). The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 1 min, 58℃ annealing for 30 s, 72℃ extension for 5 min 20 s, 34 cycles, with the final round incubated at 72℃ for 5 min, and finally cooled at 16℃ for 10 min. The PCR products were examined by agarose gel electrophoresis, and the purified gel was recovered and stored for later use. The results are shown in the figure. Figure 2 .

[0029] The above-obtained contents deoC The gel recovery product of the gene was homologously recombinated with the vector pET-28a, which had been linearized by inverse PCR, and transformed into E. coli Top10 competent cells. Positive clones were identified by PCR amplification. Figure 3 ), select transformants that have been verified by PCR, expand culture, and extract recombinant plasmid pET-28a(+)- deoC And it was verified by sequencing to be correct.

[0030] The obtained plasmid pET-28a(+)- deoC The recombinant E. coli was transformed into E. coli BL21 competent cells and inoculated into LB medium containing 50 μg / mL kanamycin sulfate, and cultured overnight at 37°C and 200 rpm with shaking. The inoculum was then transferred at a 1:50 ratio to 200 mL Erlenmeyer flasks containing LB medium and cultured at 37°C and 200 rpm with shaking. When the OD of the culture medium... 595 When the saturation point (nm) reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L as an inducer. After induction at 25°C for 15 hours, the culture medium was centrifuged, cells were collected, and washed twice with physiological saline to obtain resting cells. The obtained resting cells were resuspended in phosphate buffer (pH 7.4) and disrupted using an ultrasonic cell disruptor. After centrifugation at 12000×g for 20 min, the supernatant obtained was the crude enzyme solution of DeoC recombinase. Figure 4 The results are shown in the figure by polyacrylamide gel electrophoresis (each lane in the figure represents: 1. Protein Marker; 2. Total protein of uninduced bacterial cells; 3. Total protein after induced bacterial cell lysis; 4. Supernatant of induced bacterial cell lysis solution after centrifugation; 5. Precipitate of induced bacterial cell lysis solution after centrifugation).

[0031] The crude DeoC recombinase solution was loaded onto a nickel column. First, impurities were eluted with solution A (pH 8.0), then the target protein DeoC was eluted with solutions B and C (pH 8.0). The purified target protein was collected, and 80% glycerol was added. The solution was stored at -80°C for later use. Solution A was phosphate buffer (pH 7.4) containing 10 mM imidazole; solution B was phosphate buffer (pH 7.4) containing 100 mM imidazole; and solution C was phosphate buffer (pH 7.4) containing 500 mM imidazole. Figure 5 This is a diagram showing the purification of the recombinant protein DeoC by nickel column affinity chromatography.

[0032] Example 2: Preparation of statin drug intermediates via DeoC-catalyzed continuous aldol condensation reaction of acetaldehyde and chloroacetaldehyde. 3R , 5S )-6-chloro-2,4,6-trideoxypyranoside.

[0033] The crude DeoC enzyme solution prepared in Example 1 was mixed with phosphate buffer (pH 7.4), and substrates acetaldehyde and chloroacetaldehyde were added. The concentration of chloroacetaldehyde was 25 mM, and the molar concentration of acetaldehyde was twice that of chloroacetaldehyde. The reaction solution was thoroughly mixed and reacted with shaking at 30°C in the dark for 15 h. After the reaction, the mixture was extracted three times with ethyl acetate, and the extracts were combined and dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain (…). 3R , 5S )-6-chloro-2,4,6-trideoxypyranoside, the product was confirmed to be correct by high-resolution mass spectrometry. Figure 6 ).

[0034] The activity of the aldolase DeoC described in this invention is determined using the 2,4-dinitrophenylhydrazine colorimetric method. By detecting changes in the concentration of the substrate chloroacetaldehyde in the reaction solution, the amount of chloroacetaldehyde consumed indirectly reflects the catalytic activity of DeoC, thus providing a preliminary indication of whether an aldol reaction has occurred. Chloroacetaldehyde reacts with 2,4-dinitrophenylhydrazine to generate 2,4-dinitrophenylhydrazone. When 2,4-dinitrophenylhydrazone is placed in an alkaline environment, it generates a colored quinone compound under the action of hydroxide ions. The absorbance of the quinone compound is detected at a wavelength of 528 nm using a microplate reader, thereby calculating the amount of chloroacetaldehyde consumed from the start to the end of the reaction. This reaction is carried out at 30°C in the dark. The reaction system consists of chloroacetaldehyde, acetaldehyde, phosphate buffer (pH 7.4), and an appropriate amount of purified DeoC enzyme, and the reaction time is 1 h. At the start and end of the reaction, take 4 µL of the reaction solution and dilute it 500 times with deionized water. Take 1.6 mL of the diluted solution and place it in a 5 mL centrifuge tube. Add 300 µL of 0.1% 2,4-dinitrophenylhydrazine solution, mix well, and incubate at 28°C in the dark for 20 min. Next, add 100 µL of 100 g / L KOH solution to the mixture, mix quickly, and incubate at 28°C in the dark for 10 min. After the reaction, add 200 µL of the reaction solution to a 96-well plate and measure the absorbance at 528 nm using a microplate reader.

[0035] Example 3: Determination of DeoC activity in different pH buffers.

[0036] At a reaction temperature of 30℃, an appropriate amount of purified DeoC enzyme was added. Using 25 mM chloroacetaldehyde and 50 mM acetaldehyde as substrates, the relative activity of DeoC in buffer solutions with different pH values ​​was investigated based on changes in absorbance at a detection wavelength of 528 nm. The buffer systems used were: sodium citrate buffer (pH 4.0-6.0); phosphate buffer (pH 6.0-8.0); and Tris-HCl buffer (pH 8.0-9.0). The results are as follows: Figure 7 As shown, the optimal pH for DeoC is 7.0.

[0037] Example 4: Stability of DeoC in different pH buffers.

[0038] DeoC was incubated for 8 hours in different pH buffer environments, and the differences in DeoC enzyme activity were then detected and compared. The experimental method was the same as in Example 3, thereby comparing the stability of DeoC under different pH environments. The results are as follows: Figure 8 As shown, DeoC exhibits the best stability at pH 6.

[0039] Example 5: Activity determination of DeoC at different temperatures.

[0040] Under different temperature conditions (20℃, 30℃, 40℃, 50℃), appropriate amounts of purified DeoC enzyme were added. Using phosphate (pH 7.0) as buffer and 25 mM chloroacetaldehyde and 50 mM acetaldehyde as substrates for enzyme activity assay, the relative activity of DeoC at different temperatures was investigated based on changes in absorbance at a detection wavelength of 528 nm. Results are as follows: Figure 9 As shown, DeoC exhibits the highest catalytic activity at 30℃; as the temperature continues to rise, the enzyme activity begins to decline.

[0041] Example 6: Stability of DeoC at different temperatures.

[0042] DeoC was incubated at different temperatures (20℃, 30℃, 40℃, 50℃) for 8 h, and the differences in DeoC enzyme activity were then detected and compared. The experimental method was the same as in Example 5, thereby comparing the stability of DeoC at different temperatures. The results are as follows: Figure 10 As shown, DeoC exhibits the highest stability at 20℃.

[0043] Example 7: Determination of DeoC activity at different substrate concentrations.

[0044] Using phosphate buffer (pH 7.0), chloroacetaldehyde was added at molar concentrations of 5 mM, 10 mM, 15 mM, 25 mM, 50 mM, 75 mM, and 100 mM, with acetaldehyde at twice the molar concentration of chloroacetaldehyde. Simultaneously, an appropriate amount of purified DeoC enzyme was added, and the mixture was stirred at 30°C. The relative activity of DeoC at different substrate concentrations was investigated based on the change in absorbance at a detection wavelength of 528 nm. The results are as follows: Figure 11 As shown, DeoC exhibits the highest catalytic activity at chloroacetaldehyde concentrations of 25 mM and acetaldehyde concentrations of 50 mM. When the chloroacetaldehyde concentration is gradually increased to 100 mM, the relative activity of the enzyme remains at around 50%, indicating that the aldolase DeoC has high aldehyde tolerance to chloroacetaldehyde.

[0045] Under the optimized reaction conditions determined above, 0.1 mg of aldolase was added to catalyze the substrate reaction, and the conversion rate of the substrate chloroacetaldehyde was detected. The conversion rate was calculated using the formula: Conversion Rate = (Amount of substrate consumed in the reaction / Total initial substrate) × 100%, and the results showed that the conversion rate reached 44.45%.

[0046] The enzyme activities in the above examples were compared using the conversion rate of chloroacetaldehyde, and the standard curve was calculated as the relationship between chloroacetaldehyde concentration and OD. 528 linear relationship of nm ( Figure 12 ).

[0047] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A 2-deoxyribose-5-phosphate aldolase derived from Rhodococcus SD3, characterized in that, The amino acid sequence of this enzyme is shown in SEQ ID NO.

1.

2. A gene encoding 2-deoxyribose-5-phosphate aldolase derived from Rhodococcus SD3, characterized in that, The nucleotide sequence of this gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The vector contains the gene described in claim 2, and uses pET-28a(+) as the vector.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria comprises the recombinant expression vector of claim 3, and the host cell of the genetically engineered bacteria is Escherichia coli BL21(DE3).

5. A method for producing 2-deoxyribose-5-phosphate aldolase, characterized in that: a) Under suitable expression conditions, culture the whole cells of the genetically engineered bacteria as described in claim 4; b) Alternatively, isolate and purify the 2-deoxyribose-5-phosphoaldolase as described in claim 1 from the culture.

6. The use of the 2-deoxyribose-5-phosphate aldolase as described in claim 1 in the catalytic synthesis of (3R,5S)-6-chloro-2,4,6-trideoxypyranoside.

7. A method for synthesizing (3R,5S)-6-chloro-2,4,6-trideoxypyranoside, characterized in that, A hydrologenic condensation reaction is carried out using acetaldehyde and chloroacetaldehyde as substrates, catalyzed by the 2-deoxyribose-5-phosphoaldolase as described in claim 1. The catalytic conditions for the reaction satisfy one or more of the following: a) The reaction is carried out in a buffer system with a pH of 4.0–9.0; b) The reaction temperature is 20°C to 50°C; c) The concentration of chloroacetaldehyde is from 5 mM to 100 mM; d) The molar ratio of acetaldehyde to chloroacetaldehyde is 2:1.

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